Nucleic acid hybridization assays are commonly used in genetic research, biomedical research and clinical diagnostics. In a basic nucleic acid hybridization assay, single-stranded analyte nucleic acid is hybridized to a labeled single-stranded nucleic acid probe and resulting labeled duplexes are detected. Variations of this basic scheme have been developed to enhance accuracy, facilitate the separation of the duplexes to be detected from extraneous materials, and/or amplify the signal that is detected.
The present invention is directed to a method of reducing background noise encountered in any nucleic acid hybridization assay. Generally, the background noise which is addressed by way of the presently disclosed techniques results from undesirable interaction of various polynucleotide components that are used in a given assay, i.e., interaction which gives rise to a signal which does not correspond to the presence or quantity of analyte. The invention is useful in conjunction with any number of assay formats wherein multiple hybridization steps are carried out to produce a detectable signal which correlates with the presence or quantity of a polynucleotide analyte.
One such assay is described in detail in commonly assigned U.S. Pat. No. 4,868,105 to Urdea et al., the disclosure of which is incorporated herein by reference. That assay involves the use of a two-part capturing system designed to bind the polynucleotide analyte to a solid support, and a two-part labeling system designed to bind a detectable label to the polynucleotide analyte to be detected or quantitated. The two-part capture system involves the use of capture probes bound to a solid support and capture extender molecules which hybridize both to a segment of the capture probes and to a segment of the polynucleotide analyte. The two-part labelling system involves the use of label extender molecules which hybridize to a segment of the polynucleotide analyte, and labeled probes which hybridize to the label extender molecules and contain or bind to a detectable label. An advantage of such a system is that a plurality of hybridization steps must occur in order for label to be detected in a manner that correlates with the presence of the analyte, insofar as two distinct hybridization reactions must occur for analyte "capture," and, similarly, two distinct hybridization reactions must occur for analyte labelling. However, there remain a number of ways in which a detectable signal can be generated in a manner which does not correspond to the presence or quantity of analyte, and these will be discussed in detail below.
Another example of an assay with which the present invention is useful is a signal amplification method which is described in commonly assigned U.S. Pat. No. 5,124,246 to Urdea et al., the disclosure of which is incorporated herein by reference. In that method, the signal is amplified through the use of amplification multimers, polynucleotides which are constructed so as to contain a first segment that hybridizes specifically to the label extenders, and a multiplicity of identical second segments that hybridize specifically to a labeled probe. The degree of amplification is theoretically proportional to the number of iterations of the second segment. The multimers may be either linear or branched. Branched multimers may be in the shape of a fork or a comb, with comb-type multimers preferred.
One approach to solving the problem of interfering background signals in nucleic acid hybridization assays is provided in commonly assigned U.S. patent application Ser. No. 08/164,388 to Urdea et al. in which at least two capture extenders and/or two or more label extenders must bind to the analyte in order to trigger a detectable signal. To further reduce background noise, the assay is conducted under conditions which favor the formation of multicomponent complexes.
Another approach which has been proposed to increase the target dependence of the signal in a hybridization assay is described in European Patent Publication No. 70,685, inventors Heller et al. That reference describes a homogeneous hybridization assay in which a nonradiative transfer of energy occurs between proximal probes; two distinct events must occur for a target-generated signal to be produced, enhancing the accuracy of detection.
The present invention is also designed to increase the accuracy of detection and quantitation of polynucleotide analytes in hybridization assays. The invention increases both the sensitivity and specificity of such assays, by reducing the incidence of signal generation that occurs in the absence of target, and does not involve an increase in either time or cost relative to currently used assay configurations.
The goals of the present invention, namely to reduce background noise and to increase accuracy of detection and quantitation of analytes in nucleic acid hybridization assays have been achieved, in part, by the use of nucleoside variants that form base pairs by virtue of "non-natural" hydrogen bonding patterns. As used herein, a "non-natural" base pair is one formed between nucleotidic units other than adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine (U). One such non-natural nucleoside base pair is formed between isocytosine (isoC) and isoguanine (isoG). IsoC and isoG can form a base pair with a standard geometry (i.e., a "Watson-Crick base pair") but involving hydrogen bonding other than that involved in the bonding of cytosine (C) to guanine (G), as shown below: ##STR1##
Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 applied molecular mechanics, molecular dynamics and free energy perturbation calculations to study the structure and stability of the isoC*isoG base pair. Tor et al. (1993) J. Am. Chem. Soc. 115:4461-4467 describe a method whereby a modified isoc in a DNA template will direct the incorporation of an isoG analog into the transcribed RNA product. Switzer et al. (1993) Biochemistry 32:10489-10496 studied the conditions under which the base pair formed between isoC and isoG might be incorporated into DNA and RNA by DNA and RNA polymerases.
Introduction of a new base pair into DNA oligomers offers the potential of allowing more precise control over hybridization.